Increases in middle ear pressure resulting from counter-diffusion of oxygen and carbon dioxide into the middle ear of monkeys

Bryodin 1 (BD1) is a potent ribosome-inactivating protein (RIP) isolated from the plant Bryonia dioica. It is relatively nontoxic in rodents (LD50 > 40 mg/kg) and represents a potential improvement over other RIPs and bacterial toxins that have been used in immunotoxins. Recombinant BD1, expressed in Escherichia coli, localizes to insoluble inclusion bodies necessitating denaturation and refolding steps to generate active protein. In this report, BD1 was expressed as a soluble recombinant protein in tobacco cell culture (ntBD1) and purified to near homogeneity with yields of up to 30 mg/(L of culture). The protein synthesis inhibition activity of ntBD1 was identical to that of both native BD1 isolated from the roots of B. dioica and recombinant BD1 expressed in E. coli. Toxicology analysis showed that ntBD1 was well tolerated in rats at doses that cannot be achieved with most other toxin components of immunotoxins. Additionally, a single-chain immunotoxin composed of BD1 fused to the single-chain Fv region of the anti-CD40 antibody G28-5 (ntBD1-G28-5 sFv) was expressed in tobacco tissue culture as a soluble protein and was specifically cytotoxic toward CD40 expressing non-Hodgkin's lymphoma cells in vitro. These data indicate that tobacco tissue culture is a viable system for soluble expression of BD1 and BD1-containing immunotoxins.